發(fā)布時間：2018-06-20 15:24

  本文選題：葡萄糖 + 破骨細(xì)胞　； 參考：《吉林大學(xué)》2014年碩士論文

【摘要】：糖尿病是一種與高血糖密切相關(guān)的慢性內(nèi)分泌性疾病，可以導(dǎo)致包括骨量減少、骨折及骨質(zhì)疏松癥在內(nèi)的多種并發(fā)癥。但糖尿病性骨質(zhì)疏松相關(guān)的生物學(xué)機(jī)制仍不十分明確。近年來針對糖尿病性骨質(zhì)疏松的研究已經(jīng)成為國內(nèi)外的研究熱點(diǎn)。高血糖是引起骨穩(wěn)態(tài)平衡失調(diào)的主要因素之一,長期高血糖可產(chǎn)生大量糖基化終末產(chǎn)物(Advanced Glycosylate End Products,AGE)。高濃度的AGE可使破骨細(xì)胞酸性磷酸酶(Acid phosphatase, ACP)與抗酒石酸酸性磷酸酶(TartrateResistant Acid Phosphatase, TRAP)活性顯著增高,但葡萄糖直接對破骨細(xì)胞的影響目前尚未見報(bào)道。破骨細(xì)胞是參與骨穩(wěn)態(tài)調(diào)節(jié)的主要細(xì)胞,破骨細(xì)胞的骨吸收功能與成骨細(xì)胞的骨形成功能相互作用調(diào)節(jié),形成一種特殊的耦聯(lián)機(jī)制,維持骨穩(wěn)態(tài)。因此,該耦聯(lián)的分子機(jī)制成為近年來骨生理和骨疾病研究治療及藥物研發(fā)的重點(diǎn)對象。當(dāng)骨穩(wěn)態(tài)失調(diào)時，可以導(dǎo)致包括骨硬化、骨質(zhì)疏松等骨量異常所引發(fā)的疾病。早期關(guān)于該藕聯(lián)的分子機(jī)制研究最多的就是1997年由美日學(xué)者同時發(fā)現(xiàn)的RANKL/RANK/OPG信號系統(tǒng)。近幾年,研究者發(fā)現(xiàn)Eph/ephrin雙向信號轉(zhuǎn)導(dǎo)通路通過調(diào)節(jié)成骨細(xì)胞和破骨細(xì)胞的功能,可以同時促進(jìn)骨吸收抑制骨形成，，也可以同時促進(jìn)骨形成抑制骨吸收。這是由于ephrin配體同時兼具受體樣功能，從而產(chǎn)生雙向信號轉(zhuǎn)導(dǎo)。其中,膜蛋白Eph與配體ephrin結(jié)合，向細(xì)胞胞內(nèi)轉(zhuǎn)導(dǎo)的信號,稱之為正向信號; ephrin自身表達(dá)細(xì)胞胞內(nèi)轉(zhuǎn)導(dǎo)的信號,稱之為逆向信號。該通路憑借對骨穩(wěn)態(tài)調(diào)節(jié)功能中關(guān)鍵問題的成功詮釋,在骨組織領(lǐng)域受到越來越多的關(guān)注。 目的：本實(shí)驗(yàn)用RANKL誘導(dǎo)小鼠單核巨噬細(xì)胞RAW264.7分化為破骨細(xì)胞,同時給予不同濃度的葡萄糖干預(yù),觀察葡萄糖對破骨細(xì)胞分化及功能的影響,為糖尿病性骨病的發(fā)病機(jī)制提供進(jìn)一步的理論依據(jù)。 方法：不同葡萄糖濃度（5mmol/L、15mmol/L、30mmol/L）條件下，RANKL刺激小鼠單核巨噬細(xì)胞RAW264.7細(xì)胞，誘導(dǎo)其向破骨細(xì)胞分化。以5mmol/L為低糖對照組,15mmol/L、30mmol/L為實(shí)驗(yàn)組，以高濃度甘露醇為高滲對照組,誘導(dǎo)培養(yǎng)5天。用TRAP染色試劑盒分析各組細(xì)胞的抗酒石酸酸性磷酸酶活性變化；用Real-time PCR技術(shù)分析不同時間點(diǎn)各組細(xì)胞中破骨細(xì)胞標(biāo)志蛋白ACP5、ctsk基因的表達(dá)的變化及破骨細(xì)胞Eph/ephrin信號通路中EphA2基因及EphA2/ephrinA2信號通路下游轉(zhuǎn)錄因子c-Fos、NFATc1基因的表達(dá)變化；RAW264.7細(xì)胞與骨片共培養(yǎng)，甲苯胺藍(lán)染色后計(jì)算骨吸收陷窩數(shù)，根據(jù)骨陷窩數(shù)評估破骨細(xì)胞骨吸收功能。 結(jié)果：培養(yǎng)1天、3天、5天后采用Real-time PCR檢測ACP5、ctsk、c-Fos、NFATc1、EphA2等基因表達(dá)變化。第1、3天時，c-Fos、NFATc1表達(dá)量實(shí)驗(yàn)組明顯高于低糖對照組（P＜0.05）與高滲對照組（P＜0.05），第5天時，c-Fos、NFATc1表達(dá)量實(shí)驗(yàn)組明顯低于低糖對照組（P＜0.05）與高滲對照組（P＜0.05），且表達(dá)量隨葡萄糖濃度增加逐漸降低。第1、3、5天時，EphA2表達(dá)量實(shí)驗(yàn)組明顯低于低糖對照組（P＜0.05）與高滲對照組（P＜0.05），且表達(dá)量隨葡萄糖濃度增加逐漸降低。第1、3天時，ACP5、ctsk表達(dá)量實(shí)驗(yàn)組與對照組相比均無明顯變化，第5天時，ACP5、ctsk表達(dá)量實(shí)驗(yàn)組明顯低于低糖對照組（P＜0.05）與高滲對照組（P＜0.05），且表達(dá)量隨葡萄糖濃度增加逐漸降低。骨吸收陷窩經(jīng)甲苯胺藍(lán)染色后呈藍(lán)色,骨吸收陷窩呈圓形、橢圓形或不規(guī)則形。培養(yǎng)5天時,骨吸收陷窩數(shù)實(shí)驗(yàn)組明顯低于低糖對照組（P＜0.05）與高滲對照組（P＜0.05），且骨吸收陷窩數(shù)隨葡萄糖濃度增加逐漸降低。 結(jié)論：高濃度葡萄糖條件下，RANKL誘導(dǎo)的RAW264.7細(xì)胞分化能力下降，破骨細(xì)胞骨吸收能力降低。
[Abstract]:Diabetes is a chronic endocrine disease closely related to hyperglycemia, which can lead to a variety of complications, including bone loss, fracture and osteoporosis. However, the biological mechanism of diabetes related osteoporosis is still not very clear. Research on diabetes osteoporosis has become a domestic and foreign research in recent years. Hyperglycemia is one of the main factors causing the imbalance of homeostasis of bone. Long term hyperglycemia can produce a large number of Advanced Glycosylate End Products, AGE. High concentration of AGE can make osteoclast acid phosphatase (Acid phosphatase, ACP) and anti alcohol acid acid phosphatase (TartrateResistant Acid Phosphatase). AP) activity significantly increased, but the effect of glucose directly on osteoclasts has not yet been reported. Osteoclasts are the main cells involved in the regulation of bone homeostasis. The bone resorption function of osteoclasts can interact with the bone formation of osteoblasts, forming a special coupling mechanism to maintain bone homeostasis. Therefore, the coupled molecular machine is used. The system has become a key object in the research and development of bone physiology and bone disease in recent years. When the bone homeostasis is dysfunctional, it can lead to diseases such as bone sclerosis and osteoporosis, and the molecular mechanism of this coupling is the most important in the RANKL/RANK/OPG signal system found at the same time in 1997 by American and Japanese scholars. In recent years, researchers have found that the Eph/ephrin bi-directional signal transduction pathway can promote bone absorption and bone formation by regulating osteoblast and osteoclast, which can also promote bone formation and inhibit bone absorption. This is due to the simultaneous receptor like function of the ephrin ligand, which produces bi-directional signal transduction, including membrane protein Eph. In conjunction with the ligand ephrin, signals transduced into cell cells are called positive signals; ephrin itself expresses cell intracellular transduction signals, known as reverse signals. This pathway has attracted more and more attention in the field of bone tissue by the successful interpretation of key problems in the regulation of bone homeostasis.
Objective: in this experiment, the mouse monocyte macrophage RAW264.7 was induced to differentiate into osteoclast by RANKL, and the effects of glucose on the differentiation and function of osteoclast were observed at the same time, and further theoretical basis was provided for the pathogenesis of diabetic osteopathy.
Methods: under the condition of different glucose concentrations (5mmol/L, 15mmol/L, 30mmol/L), RANKL stimulated the mouse mononuclear macrophage RAW264.7 cells and induced their differentiation into osteoclasts. 5mmol/L was the low sugar control group, 15mmol/L, 30mmol/L as the experimental group, and the high concentration mannitol was used as hypertonic control group for 5 days. The groups were analyzed with TRAP staining kit. Changes in the activity of anti tartaric acid acid phosphatase in cells; the changes in the expression of the osteoclast marker protein ACP5, the ctsk gene in different time points and the EphA2 gene and the downstream transcription factor c-Fos of the EphA2/ephrinA2 signal pathway in the Eph/ephrin signaling pathway of osteoclasts and the expression changes of the NFATc1 gene in the cells of different time points using Real-time PCR technique RAW264.7 cells were co cultured with bone fragments, toluidine blue staining was used to calculate the number of bone resorption lacunae, and the osteoclastic bone resorption function was assessed according to the number of bone lacuna.
Results: the expression of ACP5, ctsk, c-Fos, NFATc1, EphA2 was detected by Real-time PCR for 1 days, 3 days and 5 days. At day 1,3, the experimental group of c-Fos and NFATc1 expression was significantly higher than that in the low sugar control group (P < 0.05) and the hypertonic control group (P < 0.05), and the experimental group was significantly lower than the low sugar control group at the time of fifth days. In the hypertonic control group (P < 0.05), the expression level gradually decreased with the increase of glucose concentration. At the 1,3,5 day, the expression of EphA2 in the experimental group was significantly lower than that in the low sugar control group (P < 0.05) and the hypertonic control group (P < 0.05), and the expression amount decreased gradually with the increase of glucose concentration. At the time of 1,3, the experimental group of ACP5 and ctsk expression was not obvious compared with the control group. At fifth days, the expression of ACP5 and ctsk in the experimental group was significantly lower than that in the low sugar control group (P < 0.05) and the hypertonic control group (P < 0.05), and the expression amount decreased gradually with the increase of glucose concentration. The bone resorption lacuna was blue after toluidine blue staining, and the bone resorption lacuna was round, oval or irregular. The number of resorption lacunae in the 5 days of culture was tested. The group was significantly lower than that of the low sugar control group (P < 0.05) and hypertonic control group (P < 0.05), and the number of bone resorption lacunae gradually decreased with the increase of glucose concentration.
Conclusion: under high glucose condition, the differentiation ability of RAW264.7 cells induced by RANKL decreased, and the osteoclastic bone resorption ability of osteoclasts decreased.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R587.1;R783.6

【共引文獻(xiàn)】

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相關(guān)博士學(xué)位論文 前2條

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